We have demonstrated that ERP is localized to the mitochondria in a variety of cell types and this mitochondrial localization does not change in response to ligand or to insult. We have shown that in the mitochondria, ER(3 in localized primarily in the matrix, with some associated with the inner mitochondrial membran. Further, we have recently constitutively transfected HT-22 immortalized hippocampal cells with a siRNA construct that reduced total and mitochondrial ERP by >90%. These cells are resistant to a variety of pro-oxidant or metabolic stresses. For example, they are resistant to cell death induced by H2O2, glutamate and iodoacetic acid. Further their mitochondria are resistant to H202-induced membrane potential (Anjm) collapse and they are able to maintain ATP production in spite of insults that compromise ATP production. Finally, we have recently shown that these cells are resistant to respiration increase induced by a mitochondrial uncoupling agent. In brief, our preliminary data suggest that unliganded mitochondrial ERP increases neuronal vulnerability of cells to insults. The overall goal of the present application is to determine the mechanisms by which mitochondria ERp affects cell vulnerability to insults. This goal would be achieved by pursuing 5 specific aims. Specific Aim 1 will determine the phenotype of primary neurons and transformed neuronal cells with ERp knockdown or knockout. An AAV-2 ERD-GFP construct that we have shown effectively infects nearly all primary neurons and reduces ERD will be used in these studies. Specific Aim 2 will determine the effects of over-expression of ERP using constructs that has a mitochondrial localization sequence as well as constructs that lack this sequence on the phenotype of primary mouse cortical neurons as well as HT-22 cells. Specific Aim 3 will determine the mitochondrial proteins that associate with ERp in the mouse brain. We will select candidate proteins that are already known or suspected to be affected by estrogens and using immunoprecipitation with anti-ERp and subsequent immunoblotting for the candidate proteins. Specific Aim 4 will determine if ligand binding to mitochondrial ERP changes its mitochondrial localization or its interaction with associated proteins. And finally, specific Aim 5 will determine if ERp knockout mice are resistant to neurotoxic stresses. These studies are of critical importance in our understanding of the biology of ERs, but are particularly relevant in view of the recently published WHIMS results and the resulting decline in use of estrogen and/or hormone therapy by vast numbers of women. We believe that non-use of postmenopausal estrogens produces a state of "unliganded mitochondrial ERP" that we propose increases neuronal vulnerability to insults.